Cyclic peptides are polypeptide chains taking cyclic ring structure and are known to have multiple biological activities, such as antibacterial activity, immunosuppressive activity or anti-tumor activity. Several cyclic peptides found in nature are used in the clinic such as the anti-bacterials gramicidin S, tyrocidine, and vancomycin, or cyclosporine A having immunosuppressive activity. Encouraged by natural cyclic peptides with biological activity, efforts have been made to develop artificial cyclic peptides with both genetic and synthetic methods.
An emerging class of biomolecules having cyclic structure are ribosomally synthesized peptides, which require extensive post translational modification to form the biologically active peptide. Most ribosomally synthesized natural peptides are translated as precursors composed of a leader- and a core-peptide. The leader serves as recognition sequence and recruits the enzymatic machinery to install post-translational modifications (PTMs) at specific residues of the core peptide.
Thereby the post-translational modifications, such as hetero- or macrocyclization, dehydration, acetylation, glycosylation, halogenation, prenylation, and epimerization not only emerge the biological activity of such peptides but also directly contribute to the excellent stability found in many representative members of this peptide class, and thus makes them attractive candidates for drug development.
Lanthipeptides and lantibiotics form a group of unique ribosomally synthesised and post-translationally modified antibiotic peptides that are produced by, and primarily act on, Gram-positive bacteria (for review see Knerr and van der Donk, Annu. Rev. Biochem. 2012. 81:479-505). Natural lantibiotics, such as e.g. nisin or subtilin are well studied and commercially used in the food industry for making and preserving dairy products such as cheese.
Lanthipeptides and lantibiotics as a subclass of peptides with antimicrobial activity, contain intramolecular thioether-bridges or rings formed by the thioether amino acids lanthionine (Lan) and 3-methyllanthionine (MeLan) which protect such peptides against proteolytic degradation and confer increased thermostability. Thioether-bridge installment starts with the enzymatic dehydration of serine or threonine to the unsaturated dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by intramolecular Michael-type addition of cysteine thiols and are mediated by lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). In class I lanthipeptides, serine/threonine dehydration and subsequent cyclisation are performed by a LanB type dehydratase and a LanC type cyclase, respectively, whereas in class II lanthipeptides a single bifunctional LanM type enzyme performs both reactions. Interestingly, unsaturated Dha has a high chemical reactivity and can, under mild basic conditions, readily react with the side of cysteine or lysine to yield non-stereoselective thioether-bridges and lysinoalanine-bridges, respectively. The biosynthesis of class III and class IV lanthipeptides is supported by multifunctional LanKC and LanL type enzymes, respectively, which are characterized by an amino-terminal phospho-Ser/phospho-Thr lyase domain, a central kinase-like domain, and a carboxy-terminal LanC-like domain (cyclase) (van der Donk et al. 2014 Current Opinion in Structural Biology 2014, 29:58-66).
Over the last years the principle of lanthipeptide biosynthesis was more and more adapted for the discovery and generation of artificial bioactive peptides having cyclic structure.
First in 2004 it has been proposed that the lanthipeptide-synthesizing enzymes can be advantageously used to introduce PTMs, such as thioether-bridges, into peptides that are normally unmodified, to improve the stability of the peptide and/or to alter its activity (Kuipers et al. 2004. J. Biol. Chem. 279, 22176-22182). In WO2006/062398 it was shown that a peptide of interest can be dehydrated in a host cell by an isolated lantibiotic dehydratase, such as LanB, which is not part of the conventional lantibiotic enzyme complex. It was further demonstrated that modified thioether-bridge containing peptides can be secreted by a protein export system other than the dedicated lantibiotic transporters in its natural host.
Later, in WO2012/005578 it was demonstrated that thioether-bridge containing peptides could readily be produced by, and displayed on, the surface of a host cell (e.g. Lactococcus lactis) which expresses the biosynthetic and export machinery for lantibiotics.
More specifically, WO2012/005578 provides an expression vector encoding a fusion peptide comprising an N-terminal lantibiotic leader sequence, an amino acid sequence of interest to be post-translationally modified to a dehydroresidue- or thioether-containing polypeptide and a C-terminal charged membrane anchoring domain. Also a display library to screen for cyclized peptides with a desired activity was suggested. However, only display on Gram-positive host cells, in particular lactic acid bacteria, which by nature are able to produce lantibiotics was enabled.
Other display systems known in the art such as phage display that require gram-negative bacteria having a different protein export machinery were not considered as an alternative and therefore were not enabled in the prior art.
The story of phage display started in 1985 based on the demonstration that filamentous phage tolerate foreign protein fragments inserted in their gene III protein (pIII) and also present the protein fragments on the phage surface (Smith, 1985). Ladner extended that concept to the screening of repertoires of (poly)peptides and/or proteins displayed on the surface of phage (WO1988/06630; WO1990/02809) and, since then, phage display has experienced a dramatic progress and resulted in substantial achievements. Various formats have been developed to construct and screen (poly)peptide/protein phage-display libraries, and a large number of review articles and monographs cover and summarise these developments (e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996). To anchor the peptide or protein to the filamentous bacteriophage surface, mostly genetic fusions to phage coat proteins are employed. Preferred are fusions to gene III protein (Parmley & Smith, 1988) or fragments thereof (Bass et al., 1990), and gene VIII protein (Greenwood et al., 1991). In one case, gene VI has been used (Jespers et al., 1995), and recently, a combination of gene VII and gene IX has been used for the display of Fv fragments (Gao et al., 1999).
So far only linear (poly) peptides and cyclic peptides stabilized via disulphide bonds were successfully displayed on phages (see WO2000/077194, WO2009/098450). More recently in WO2012/019928 the linear precursor of Microvividrin K fused to the N-terminus of the pIII was displayed on phage. The post-translational modification of the displayed linear precursor was achieved by subsequent incubation of the phages with cell lysates containing the cognate modifying enzymes. However, WO2012/019928 does not provide enabling disclosure for the display of lanthipeptides and also does not teach the display of peptides which underwent post-translational modification prior to phage assembly.
Accordingly, a need exists to translate the display of cyclic post-translationally modified peptides from bacteria to classic phage display.